During the course of an infection process the organism develops a broad humoral response directed against different pathogen's antigens which, in conjunction with the innate and T-cell responses, control the infection and preserve the integrity of the organism. In the setting of the HIV-1 infection, this humoral response can be detected early during the infection but it is known to be ineffective, mainly because the antibodies produced by most patients recognize viral epitopes which cannot interfere with the replicative cycle of the virus. See Tomaras G, et al., J. Virol. 2008; 82:12449-12463. In fact, antibodies with the capacity to neutralize the autologous virus can be identified after few months only in some patients; and several years are required to develop broadly neutralizing antibodies (bnAbs). See Mascola J, et al., Annu. Rev. Immunol. 2010; 28:413-444. To date, only a few broadly neutralizing antibodies have been identified and all of them recognize a set of conserved epitopes in the envelope protein with an important role in viral fitness. These antibodies include anti-CD4 binding site (CD4bs) antibodies (IgGb12 and VCR01), anti-CD4 induced-epitope antibodies (X5), anti-gp41 antibodies (2F5 and 4E10), anticarbohydrates (2G12), anti-glycosylated quaternary epitopes (PG9 and PG16) and anti-core antibodies. See Barbas C, et al., Proc. Natl. Acad. Sci USA 1992; 89:9339-9343, Wu X, et al., Science 2010; 329: 856-861, Moulard M, et al., Proc. Natl. Acad. Sci. USA 2002; 99:6913-6918, Muster T, et al., J. Virol. 1993; 67:6642-6647, Zwick M, et al., J. Virol. 2001; 75:10892-10905, Scanlan C, et al., J. Virol. 2002; 76:7306-7321, Walker L, et al., Science 2009; 326:285-289, and Pietzsch J, et al., J. Exp. Med. 2010; 207:1995-2002. Among them, antibodies that can block the interaction of gp120/CD4 such as anti-CD4bs antibodies can be highlighted for several reasons: 1) they recognize a conserved region of gp120, 2) they can neutralize a broad number of viral isolates and 3) they can prevent or control the infection as it has been shown in animal models of HIV-1 infection. See Hessell A, et al., Nat. Med. 2009; 15:951-954, Hessell A, et al., Nature 2007; 449:101-104, and Veazey R, et al., Nat. Med. 2003; 9:343-346. Therefore, the elicitation of this sort of bnAbs is an interesting goal for any vaccination strategy. However, one of the major handicaps in the study of these antibodies is their identification. Broadly neutralizing antibodies in general, and CD4bs antibodies in particular, recognize conformational epitopes which are difficult to mimic in vitro. To date, several strategies have been followed to study CD4bs antibodies, including the use of recombinant proteins and mutant variants which are differentially recognized by these antibodies. See Li Y, et al., Nat. Med. 2007; 13:1032-1034, and Lynch R, et al., J. Virol. 2012; 86(4):7588-7595, and Wu, 2010, supra. In addition, a cell-to-cell viral transfer assay was recently developed which allows detecting the presence of CD4/gp120 blocking antibodies in plasma samples. This assay is based on the viral entrance process, which is completely inhibited in the presence of antibodies that block the gp120/CD4 interaction like CD4bs or anti-CD4 antibodies. By definition any antibody which is able to block the interaction between gp120 and the CD4 receptor might be a neutralizing antibody. Furthermore, this approach showed a strong correlation between the presence of gp120/CD4 blocking antibodies and the neutralizing capacity of the plasma. See Sanchez-Palomino S, et al., Vaccine 2011; 29:5250-5259. More recently, it has been shown that more than 80% of HIV-1 infected patients can develop CD4bs antibodies, indicating that this reactivity might be more frequent than it has been previously described. See Lynch, 2012, supra.
However, no clear correlation between the presence of these antibodies and the neutralizing capacity of the plasma samples could be established in this case. These discrepancies highlight that the methodology is an important issue to take into account before planning the analysis of gp120/CD4 blocking antibodies. There is a need in the art for more rapid and reliable methods for identifying HIV neutralizing antibodies.